Laboratory Testing of Genital Tract Specimens

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Laboratory Testing of Genital Tract Specimens
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Cast of Characters
Classic Modern MSM Rare in US
N. gonorrhoeae C. trachomatis Shigella H. ducreyi
T. pallidum HSV HBV LGV
Phthirus pubis HIV Campylobacter C. granulomatis
Sarcoptes scabiei T. vaginalis Giardia Molluscum contagiosum
M. hominis
U. urealyticum
M. genitalium
HPV
CMV
HCV
EBV
(Bacterial vaginosis)
(yeast vaginitis)
(Group B strep)
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The Vaginal Life Cycle
Newborn Premenarche Menstrual Postmenopause
Estrogen Yes No Yes No
Glycogen Yes No Yes No
pH lt4.5 gt4.5 lt4.5 gt4.5
Predominant Endogenous flora Lactobacillus Coag-neg staph, enterococcus, E. coli Lactobacillus G. vaginalis Coag-neg staph, enterococcus, E. coli
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Characteristics of the Vagina and Cervix in Women
of Reproductive Age
Vagina Cervix
pH lt4.5 7.0
Epithelial Cells Squamous Columnar
Pathogens/Syndromes Bacterial vaginosis Candida species Trichomonas vaginalis Neisseria gonorrhoeae Chlamydia trachomatis
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Vaginitis/Vaginosis

• Published guidelines recommend testing only for
• Bacterial vaginosis 40-50
• Yeast vaginitis 20-25
• Trichomoniasis lt5-75
• 25 of women with vaginitis have gt1

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Other organisms

• Organisms such as E. coli, Klebsiella, and
  Enterococcus are part of the endogenous flora or
  are present as fecal contaminants and do not
  cause vaginitis
• Culture for or reporting the presence of these
  organisms, e.g., is inappropriate and will lead
  to unnecessary antimicrobial therapy

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Bacterial Vaginosis

• Most common of the vaginitides
• OB clinic patients 10-26
• Other clinic populations
• 12-25
• STI clinic patients 32-64

Symptoms BV Normal
n 69 242
Present 46.4 31.8
Absent 53.6 68.2
Discharge, odor or irritation
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BV Sequelae

• OB Complications
• Preterm delivery
• Premature rupture of membranes
• Amniotic fluid infection
• Chorioamnionitis
• Postpartum endometritis
• Premature labor
• Low birth weight

• GYN Complications
• Pelvic inflammatory disease
• Postabortal pelvic inflammatory disease
• Posthysterectomy infections
• Mucopurulent cervicitis
• Endometritis
• Increased risk of HIV

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BV Clinical Characteristics

• 3 of these 4 needed for a clinical diagnosis
• vaginal fluid pH gt4.5
• homogeneous discharge
• fishy odor on addition of KOH
• clue cells

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BV Clinical Characteristics (cont.)

• Clue cells vaginal epithelial cells so coated
  with bacteria that the cell borders are
  obliterated. Were originally described on wet
  mount.
• Watch out for pseudo clue cells

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Common Endogenous Flora

• Facultative Anaerobes URT GU(F) GI Skin
• Streptococcus X 20
• Enterococcus 28 X
• Staph aureus 2-10
  X
• Lactobacillus X 100
• Gardnerella 70
• Corynebacterium 40
  X
• Enterobacteriaceae 18 X
• Neisseria/Haemophilus X

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Common EF (cont.)

• Anaerobes URT GU(F) GI
  Skin
• Bacteroides fragilis
  4-40 X
• Prevotella/Porphyromonas 12-33
• Fusobacterium X 7-28
  
• Veillonella X
  0-27
• Clostridium/Eubacterium 0-13
  X
• Actinomyces X
• Peptostreptococcus X low
  X
• Propionibacterium 0-8
  X
• CID 32e69-77, 2001

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BV-associated Flora
Normal BV
Lactobacillus High Low
Gardnerella vaginalis Varies High
Prevotella/Porphyromonas Low High
Peptostreptococcus Low High
Mobiluncus species Low High
Fusobacterium Low Higher
Mycoplasma hominis Low Higher
Atopobium vaginae Low Higher
BVAB (NEJM 3531899, 2005) Low High
Total CFU/mL 105-107 1010-1011
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Culture not recommended because

• there is no single pathogen
• is an alteration in relative numbers of EF
• 50 of women with 3-4 G. vaginalis have BV and
  the other 50 do not
• culture for associated flora is slow and costly
  some are uncultivable
• reporting the presence of Enterobacteriaceae, S.
  aureus or Enterococcus may lead to unnecessary
  antibiotic exposure
• there are better methods for laboratory diagnosis
  of BV

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Gram Stain Diagnosis

• predominance of lactobacilli gardnerella
  normal
• mixed small gram-positive and gram-negative rods
  curved rods BV
• Kopeloff modification of the Gram stain with
  basic fuchsin counterstain

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Gram Stain Diagnosis (cont.)
Score 0, no cells present 1,
lt1cell/OIF 2, 1-5 cells/OIF

3, 5-30 cells/OIF 4, gt30
cells/OIF
Interpretation 1-3, normal 4-6 intermediate
(altered vaginal flora) 7-10, BV
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Normal Vaginal Gram Stain
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NL
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Intermediate Gram Stain
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AVF
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BV
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BVM
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Affirm VPIII Instrument
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Affirm method

• a DNA probe test for T. vaginalis, Candida spp.,
  and Gardnerella vaginalis
• single swab treated to release and stabilize RNA
• capture probes on a probe analysis card
• after exposure of card to sample, exposed to
  detection probes with bound enzyme
• in the presence of enzyme substrate a blue color
  develops to indicate a positive test

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BV Other Diagnostic Methods

• Affirm VPIII
• vs. G. vaginalis culture sens 97 spec 71
• vs G. vaginalis culture and pH gt4.5 sens 94
  spec 81
• JCM 32(1)148-152, 1994

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BV Other Diagnostic Methods

• Biochemical tests (point-of-care tests)
• FemExam (Litmus Concepts) STD 30(6)483-9, 2003
• I pH and amines 71.4 sensitive, 72.8
  specific
• II proline iminopeptidase 70.0 sensitive,
  80.9 specific
• I II 91.0 sensitive, 61.5 specific
• BVBlue (Gryphus Diagnostics)
• sialidase (Bacteroides, Gardnerella, Prevotella)
• 91.7/88 sensitive, 97.8/95 specific vs. Gram
  stain
• JCM 41(5)1925-8, 2003 JCM 43(2)584-7, 2005

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Vulvovaginal Candidiasis (aka Yeast Vaginitis)

• 2nd most prevalent
• Estimated 13 million cases/year
• 70-75 1 episode in lifetime
• 40-50 2 episodes in lifetime
• 5-8 multiple episodes
• Signs and symptoms
• Curd-like discharge
• Vaginal itching (pruritus)

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Vulvovaginal Candidiasis (cont.)

• Acute
• Candida albicans (95) C. glabrata (5)
• Chronic
• C. albicans (68), C. glabrata (16),
  Saccharomyces cerevisiae (6) C. tropicalis (4)

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Diagnostic Methods

• Wet mount (KOH or calcofluor white)
• estimated at 50 sensitivity (KOH)
• False negative KOH more likely if C. glabrata.
• Affirm VPIII vs. culture
• 79 sensitive, 85 specific

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When is vaginal culture for yeast appropriate?

• Symptomatic patient
• KOH wet mount negative
• No other diagnosis
• Chromagar Candida
• If gt10 colonies, then In a symptomatic patient
  with no other genitourinary infection, treatment
  for yeast vaginitis is warranted.

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T. vaginalis vaginitis (cont.)

• Third most common STI
• 170 million annually8 million in N.A.
• Prevalence lt5-75
• Up to 50 asymptomatic
• Signs and symptoms
• Copious, greenish discharge
• Strawberry cervix
• Sequelae
• increased PID, infertility, post surgical
  infections, HIV transmission, preterm birth
• Clin Micro Rev 17(4)794-803, 2004

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T. vaginalis in men

• gt50 asymptomatic
• symptomatic infections
• urethritis (nongonococcal urethritis)
• prostatitis
• male factor infertility?

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T. vaginalis Microscopic Diagnosis

• Microscopy
• saline wet mount
• 50-60 sens in women, hi specificity
• 30 sens in men, low specificity
• Pap smear
• 50 sensitivity, 90 specificity
• low PPV in low prevalence pop

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T. vaginalis Culture Diagnosis

• In-Pouch TV
• 85-95 sens in women, gt95 spec
• 60 sens and high specificity in men (urethral
  swab or FVU sediment)

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T. vaginalis Antigen Detection

• OSOM Trichomonas Rapid Test (Genzyme Diagnostics,
  Cambridge, MA)
• 10 min point-of-care test
• 83.3 sensitive 98.8 specific vs. culture
• (wet mount 71.4 sensitive)
• JCM 43(2)664-7, 2005

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T. vaginalis Molecular Methods (cont)

• Affirm
• For Trichomonas Sensitivity 90.5 Specificity
  99.8
• PCR
• gt90 sensitive and gt95 specific for females
• gt90 sensitive and specific for males
• Culture 70 sensitive vs. PCR

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When is it appropriate to perform a bacterial
CULTURE?

• group B strep
• 35-37 wk gestation
• rectovaginal sample
• enrichment (followed by culture or AccuProbe)
• Trans-Vag broth
• LIM
• StrepB Carrot broth
• PCR
• direct
• post enrichment
• NOT direct antigen tests

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Whentoculture? (cont.)

• toxic shock syndrome
• 2-10 have S. aureus as endogenous flora
• 10 of those are TSST-1 producers
• desquamative inflammatory vaginitis (DIV)
• uncommon, purulent vaginitis
• mean age 41.8 years
• associated with Group B strep
• patients respond to clindamycin

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Do NOT culture for

• Endogenous or contaminating flora
• Gardnerella vaginalis
• Enterobacteriaceae
• E. coli
• Klebsiella species
• Proteus species
• Pseudomonas
• Enterococcus

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Whentoculture? (cont.)

• pediatric patients
• Group A strep
• Shigella species, esp. S. flexneri
• Campylobacter jejuni/coli
• Neisseria gonorrhoeae
• Chlamydia trachomatis

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Whentoculture? (cont.)

• Female upper tract infections( e.g., pelvic
  inflammatory disease (PID), salpingitis, post
  hysterectomy, post abortion)
• upper tract sample or cervical swab
• culture for known pathogens
• S. aureus, Streptococcus pyogenes (BAP, CNA)
• Neisseria gonorrhoeae (GC-lect, Choc)
• Enterobacteriaceae (EMB/MAC)
• Bacteroides fragilis, Clostridium sordellii
  (BBE/LKV, EYA)

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Sources

• www.guidelines.gov
• Management of women of reproductive age attending
  non-genitourinary medicine settings complaining
  of vaginal discharge. J Fam Plan. Reprod. Health
  Care 32(1)33-42, 2006
• Brigham and Womens Hospital. Common gynecologic
  problems a guide to diagnosis and treatment.
  Boston (MA) Brigham and Womens Hospital 2003

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Thank you!!
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Carol A. Spiegel, MT(ASCP), PhD, D(ABMM), F(AAM)

• University of Wisconsin Hospital Clinics
• 600 Highland Ave. CSC 2472
• Madison, WI 53791-3284
• ca.spiegel_at_hosp.wisc.edu